C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription
Abstract Background Acute myeloid leukemia (AML) with biallelic (CEBPA bi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible tran...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | Journal of Experimental & Clinical Cancer Research |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13046-024-02975-3 |
| _version_ | 1797258977693663232 |
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| author | Mengbao Du Mowang Wang Meng Liu Shan Fu Yu Lin Yankun Huo Jian Yu Xiaohong Yu Chong Wang Haowen Xiao Limengmeng Wang |
| author_facet | Mengbao Du Mowang Wang Meng Liu Shan Fu Yu Lin Yankun Huo Jian Yu Xiaohong Yu Chong Wang Haowen Xiao Limengmeng Wang |
| author_sort | Mengbao Du |
| collection | DOAJ |
| description | Abstract Background Acute myeloid leukemia (AML) with biallelic (CEBPA bi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs. Methods Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents. Results We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo. Conclusion Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPA bi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers. |
| first_indexed | 2024-04-24T23:02:06Z |
| format | Article |
| id | doaj.art-f2ee6830177f4cab94e60759fa6180f1 |
| institution | Directory Open Access Journal |
| issn | 1756-9966 |
| language | English |
| last_indexed | 2024-04-24T23:02:06Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
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| series | Journal of Experimental & Clinical Cancer Research |
| spelling | doaj.art-f2ee6830177f4cab94e60759fa6180f12024-03-17T12:42:12ZengBMCJournal of Experimental & Clinical Cancer Research1756-99662024-03-0143111810.1186/s13046-024-02975-3C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcriptionMengbao Du0Mowang Wang1Meng Liu2Shan Fu3Yu Lin4Yankun Huo5Jian Yu6Xiaohong Yu7Chong Wang8Haowen Xiao9Limengmeng Wang10Bone Marrow Transplantation Center of The First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of MedicineBone Marrow Transplantation Center of The First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of MedicineDepartment of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute, City of Hope Medical CenterBone Marrow Transplantation Center of The First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of MedicineBone Marrow Transplantation Center of The First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of MedicineHematology Department, The First Affiliated Hospital of Zhengzhou UniversityBone Marrow Transplantation Center of The First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of MedicineInstitute of Hematology, Zhejiang UniversityHematology Department, The First Affiliated Hospital of Zhengzhou UniversityDepartment of Hematology, Sir Run Run Shaw Hospital, Zhejiang University School of MedicineBone Marrow Transplantation Center of The First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of MedicineAbstract Background Acute myeloid leukemia (AML) with biallelic (CEBPA bi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs. Methods Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents. Results We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo. Conclusion Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPA bi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.https://doi.org/10.1186/s13046-024-02975-3CCAAT-enhancer-binding protein αCEBPA mutationAcute myeloid leukemiaDNA damage-inducible transcript 3Unfolded protein responseEndoplasmic reticulum stress |
| spellingShingle | Mengbao Du Mowang Wang Meng Liu Shan Fu Yu Lin Yankun Huo Jian Yu Xiaohong Yu Chong Wang Haowen Xiao Limengmeng Wang C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription Journal of Experimental & Clinical Cancer Research CCAAT-enhancer-binding protein α CEBPA mutation Acute myeloid leukemia DNA damage-inducible transcript 3 Unfolded protein response Endoplasmic reticulum stress |
| title | C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription |
| title_full | C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription |
| title_fullStr | C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription |
| title_full_unstemmed | C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription |
| title_short | C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription |
| title_sort | c ebpα p30 confers aml cell susceptibility to the terminal unfolded protein response and resistance to venetoclax by activating ddit3 transcription |
| topic | CCAAT-enhancer-binding protein α CEBPA mutation Acute myeloid leukemia DNA damage-inducible transcript 3 Unfolded protein response Endoplasmic reticulum stress |
| url | https://doi.org/10.1186/s13046-024-02975-3 |
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